Intratumor heterogeneity (ITH) plays a crucial role in tumor evolution, treatment response, and disease prognostication. We have developed a FISH based approach to enumerate gene and chromosomal copy number changes in single interphase nuclei (miFISH) of primary tumors and derived cell lines. This approach is based on the sequential hybridization of cancer relevant probes. The multiplex hybridization of these probes allows enumeration of up to 20 loci in individual cancer cells, therefore providing a comprehensive picture of ITH. The microscope system, automated image acquisition and signal enumeration was developed with the company BioView (Rehovot, Israel). In collaboration with Drs. Alejandro Schaeffer and E. Michael Gertz (both NCBI) and Dr. Russell Schwartz (Carnegie Mellon University, Pittsburgh, PA) we developed an algorithm that allows interpretation of the complex data sets, termed FISHtres. FISHtrees has been applied for the analysis of genome dynamics of breast, prostate, colorectal, head and neck, and cervical carcinomas, their precursor lesions, and derived cell lines.